Membrane virus host range mutations and their uses as vaccine substrates

ABSTRACT

The present invention is directed to genetically engineered, membrane-enveloped viruses with deletion mutations in the protein transmembrane domains. Also provided are viral vaccines based on the engineered viruses, methods of producing and using such vaccines.

CROSS REFERENCE TO RELATED APPLICATION

[0001] This patent application is a continuation-in-part of U.S.application Ser. No. 09/447,103, filed Nov. 22, 1999, which is acontinuation-in-part of U.S. application Ser. No. 09/157,270, filed Sep.18, 1998.

FEDERAL FUNDING LEGEND

[0002] This invention was produced in part using funds obtained througha grant from the National Institutes of Health. Consequently, thefederal government has certain rights in this invention.

BACKGROUND OF THE INVENTION

[0003] 1Field of the Invention

[0004] The present invention relates generally to virology and diseasecontrol. More specifically, the present invention relates to mutatedarthropod vectored viruses and their uses, for example, as vaccines.

[0005] 2 Description of the Related Art

[0006] Arthropod vectored viruses (Arboviruses) are viral agents whichare transmitted in nature by blood sucking insects. Arboviruses includemembers of the alpha-, flavi- and bunya-viridae. Over 600 of theseviruses are presently known and emerging members of these families arebeing described annually. Collectively, the arthropod vectored virusesare second only to malaria as a source of insect-transmitted disease anddeath in man and animals throughout the world (Berge A. O. 1975). Amongthese viral agents are Eastern, Western, and Venezuelan EquineEncephalitis Viruses, Dengue Fever, Japanese Encephalititis, San AngeloFever, West Nile Fever and Yellow Fever. Furthermore, diseases caused bythese agents are in resurgence in North America (NIAID Report of theTask Force on Microbiology and Infectious Diseases 1992, NIH PublicationNo. 92-3320) as a result of the introduction of the mosquito vectorAedes albopictus (Sprenger, and Wuithiranyagool 1985).

[0007] By their very nature, Arboviruses must be able to replicate inthe tissues of both the invertebrate insect and the mammalian host(Brown, D. T., and L. Condreay, 1986, Bowers et al. 1995). Differencesin the genetic and biochemical environment of these two host cellsystems provide a basis for the production of host range mutant viruseswhich can replicate in one host but not the other.

[0008] Currently, Dengue Fever and Eastern Equine Encephalitis and otherinsect borne viruses are in resurgence in the United States. The U.S.Army and other government agencies have been trying to make vaccinesagainst these viruses since the 1960s with little success. Thus, theprior art is deficient in a vaccine against most arthropod vectoredviruses and other membrane-coated viruses. The present inventionfulfills this long-standing need and desire in the art.

SUMMARY OF THE INVENTION

[0009] Viruses which are transmitted in nature by blood sucking insectsare a major source of disease in man and domestic animals. Many of theseviruses have lipid membrane bilayers with associated integral membraneproteins as part of their three dimensional structure. These viruses arehybrid structures in which the proteins are provided by the geneticinformation of the virus and the membrane is the product of the hostcell in which the virus is grown. Differences in the composition of themembranes of the mammalian and insect host are exploited to producevirus mutants containing deletions in the membrane spanning domains ofthe virus membrane proteins. Some of the mutants are capable ofreplicating and assembling normally in the insect host cell but assemblepoorly in the mammalian host cell. These host range mutants produceimmunity to wild type virus infection when used as a vaccine in mice,and represent a novel strategy for the production of vaccines againstarthropod vectored, membrane containing viruses.

[0010] In one embodiment of the present invention, there is provided agenetically engineered membrane-enveloped virus comprising a viraltransmembrane glycoprotein that is able to span or correctly integrateinto the membrane of insect cells but not that of mammalian cells due todeletion of one or more amino acids in the viral transmembraneglycoprotein. The virus is capable of infecting and producing progenyvirus in insect cells, and is capable of infecting but not producingprogeny virus in mammalian cells. The virus can be an Arthropod vectoredvirus such as Togaviruses, Flaviviruses, Bunya viruses and all otherenveloped viruses which can replicate naturally in both mammalian andinsect cells, as well a s enveloped viruses which can be made toreplicate in mammalian and insect cells by genetic engineering of eitherthe virus or the cell. Representative examples of such engineeredviruses are ΔK391 virus, TM17 virus and TM16 virus.

[0011] In another embodiment of the present invention, there is provideda method of producing a viral vaccine by introducing the engineeredvirus disclosed herein into insect cells and allowing the virus toreplicate in the insect cells to produce a viral vaccine. Representativeexamples of the engineered viruses are ΔK391 virus, TM17 virus and TM16virus.

[0012] In still another embodiment of the present invention, there isprovided a method for vaccinating an individual in need of suchtreatment comprising the step of introducing the viral vaccine of thepresent invention into the individual to produce viral proteins forimmune surveillance and stimulate immune system for antibody production.

[0013] In still yet another embodiment of the present invention, thereis provided a method of producing a viral vaccine to a disease spread bya wild mosquito population to mammals, comprising the steps ofengineering a deletion of one or more amino acids in a viraltransmembrane protein to produce an engineered virus similar to TM16,TM17 or delta K391, wherein the transmembrane protein is able to spanthe membrane envelope in mosquito cells but not in mammalian cells;introducing the engineered virus into the wild mosquito population; andallowing the engineered virus to replicate in cells of the wild mosquitopopulation to produce a population of mosquitoes which excludes the wildtype pathogenic virus and harbors the vaccine strain of the virus sothat a mosquito bite delivers the vaccine to the mammal bitten. Presenceof the mutated virus renders the mosquito incapable of transmittingother membrane containing viruses (Karpf et al 1997).

[0014] Other and further aspects, features, and advantages of thepresent invention will be apparent from the following description of oneof the presently preferred embodiments of the invention. Theseembodiments are given for the purpose of disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The appended drawings have been included herein so that theabove-recited features, advantages and objects of the invention willbecome clear and can be understood in detail. These drawings form a partof the specification. It is to be noted, however, that the appendeddrawings illustrate preferred embodiments of the invention and shouldnot be considered to limit the scope of the invention.

[0016]FIG. 1 shows the configuration of Sindbis virus glycoproteinsafter integration into the ER. The protein is a multipass protein with 6membrane spanning domains (numbered 1-6). 1. The signal sequence forinitial integration; 2. The first E2 transmembrane domain (TMD); 3. Thesecond E2 transmembrane domain; 4. The first 6k TMD; 5. The second 6ktransmembrane domain; and 6. The E1 transmembrane domain. S=point ofcleavage by signal peptidase.

[0017]FIG. 2 shows the results of radiolabeled Sindbis virus proteinsrecovered from transfected tissue-cultured cells. BHK-21 cells mocktransfected (1), transfected with mutant Δ391 RNA (2), and Aedesalbopictus cells transfected with Δ391 RNA (3), were labeled withradioactive amino acids as described in Example 3. At 24 hourspost-transfection, proteins were precipitated with virus specificanti-serum as described in Example 4. The figure shows that both BHK-21cells and Aedes albopictus cells transfected with RNA of the deletionmutant produce the three viral structural proteins E1, E2, and C whichare not detected in the mock transfected cells.

[0018]FIGS. 3A and 3B are electron micrographs of BHK-21 cells (FIG. 3A)and Aedes albopictus cells (FIG. 3B) transfected with RNA of the Sindbisvirus deletion mutant Δ391. Cells were transfected as described inExample 2. BHK-21 cells (FIG. 3A) show clusters of virus core structuresin the cell cytoplasm (A) even though these cells produce very lowlevels of mature virus (Table 1). Aedes albopictus cells (FIG. 3B) alsoproduce clusters of virus cores; however, these cores are found free inthe cells' cytoplasm similar to those in BHK-21 cells (A) and are alsofound associated with cell membranes (B). This latter case is not foundin BHK-21 cells, indicating that the glycoproteins E1 and E2, althoughpresent, do not function to bind them.

[0019]FIG. 4 shows the deleted amino acids in the E2 transmembranaldomain. The deleted sequence is shown under the appropriate amino acid,ranging from 1 to 16 deletions. Histidine and Proline sequencesbeginning at nt 9717 are on the lumenal side of the protein but are usedto design the mutagenic primers.

[0020]FIG. 5 shows circulating Sindbis virus antibody determined byELISA. Mutant viruses from transfected mosquito U4.4 cells were injectedinto 25 adult CD-1 mice to establish the protective index (Table 4).Injections of live mutants and UV inactivated viruses were repeated into3 additional mice to determine Ab titers by standard ELISA. The resultspresented are from a 10⁻² dilution of mouse serum.

[0021]FIG. 6 shows circulating neutralizing antibody. Antiserum used inthe experiment described in FIG. 5 was also assayed for neutralizing Ab.The neutralizing Ab data presented represent the % of wild typeinfectious virus inactivated by a 10⁻² dilution of serum from 3 adultCD-1 mice.

DETAILED DESCRIPTION OF THE INVENTION

[0022] It will be apparent to one skilled in the art that varioussubstitutions and modifications may be made to the invention disclosedherein without departing from the scope and spirit of the invention.

[0023] As used herein, the term “membrane-bound virus” refers to a viruswhich contains a lipid membrane bilayer as part of its protectiveexterior coat.

[0024] As used herein the term “viral envelope” refers to the lipidmembrane component of the membrane containing virus and its associatedproteins.

[0025] As used herein, the terms “arthropod vectored virus” or“Arbovirus” refer to viral agents which replicate and produce progenyvirus in arthropod (insect) or mammalian cells. This includesTogaviruses, Flaviviruses and Bunyaviruses.

[0026] As used herein, the term “Togavirus” refers to a generalclassification of membrane containing viruses which include theAlphaviruses.

[0027] As used herein, the term “membrane bilayer” refers to a structureconsisting of opposed amphiphatic phospholipids. The bilayer isorganized in cross section from polar head groups to non-polar carbonchains to nonpolar carbon chains to polar head groups.

[0028] As used herein, the term “glycoprotein transmembrane region”refers to the amino acid sequence of the region of a membrane-integratedprotein which spans the membrane bilayer.

[0029] As used herein, the term “viral vaccine” refers to a strain ofvirus or virus mutant which has the antigenic properties of the virusbut cannot produce disease.

[0030] As used herein the term “immune surveillance” refers to a processby which blood lymphocytes survey the cells and tissues of a mammal todetermine the presence of foreign (virus) proteins and stimulates theproduction of lymphocytes capable of targeting cells producing theforeign protein for destruction. This process also leads to theproduction of circulating antibodies against the foreign protein.

[0031] As used herein, the term “infectious virus particles” refers toviruses which are capable of entering a cell and producing virusprotein, whether or not they are capable of producing progeny virus.

[0032] As used herein, the term “non-infectious virus particles” refersto viruses which are not capable of infecting or entering a cell.

[0033] As used herein, the term “vertebrate cells” refers to anymammalian cell.

[0034] As used herein, the term “invertebrate cells” refers to anyinsect cell.

[0035] In accordance with the present invention there may b e employedconventional molecular biology, microbiology, and recombinant DNAtechniques within the skill of the art. Such techniques are explainedfully in the literature. See, e.g., Maniatis, Fritsch & Sambrook,“Molecular Cloning:” A Laboratory Manual (1982); “DNA Cloning: APractical Approach,” Volumes I and II (D. N. Glover ed. 1985);“Oligonucleotide Synthesis” (M. J. Gait ed. 1984); “Nucleic AcidHybridization” (B. D. Hames & S. J. Higgins eds. (1985)); “Transcriptionand Translation” (B. D. Hames & S. J. Higgins eds. (1984)); “Animal CellCulture” (R. I. Freshney, ed. (1986)); “Immobilized Cells And Enzymes”(IRL Press, (1986)); B. Perbal, “A Practical Guide To Molecular Cloning”(1984).

[0036] The vaccines of the present invention are based on deletionmutations in the transmembrane domains of membrane glycoproteins ofmembrane-enveloped viruses. Many membrane-coated viruses have membraneglycoproteins on their surface which are responsible for identifying andinfecting target cells (Schlesinger, S. and M. J. Schlesinger, 1990).These membrane glycoproteins have hydrophobic membrane-spanning domainswhich anchor the proteins in the membrane bilayer (Rice et al 1982).

[0037] The membrane-spanning domains of these transmembrane proteinsmust be long enough to reach from one side of the bilayer to the otherin order to hold or anchor the proteins in the membrane. Experimentshave shown that if the domains are shortened by the deletion of aminoacids within the domain, the proteins do not appropriately associatewith the membrane and fall out (Adams and Rose, 1985).

[0038] Unlike mammalian cell membranes, the membranes of insect cellscontain no cholesterol (Clayton 1964, Mitsuhashi et al 1983). Becauseinsects have no cholesterol in their membranes, the insect-generatedviral membrane will be thinner in cross section than the viral membranesgenerated from mammals. Consequently, the membrane-spanning domains ofproteins integrated into insect membranes do not need to be as long asthose integrated into the membranes of mammals. It is possible,therefore, to produce deletions in engineered viruses which remove aminoacids from the transmembrane domain of the viral glycoprotein. Thisresults in a glycoprotein which can integrate normally into the membraneof a virus replicating in an insect cell, but not into the membrane of avirus replicating in a mammal. Thus, the mutated virus can replicate andbe produced in insect cells as well as the parent wild-type virus. Onthe other hand, the mutant virus can infect mammalian cells and produceviral proteins; however, since the mutated virus glycoprotein cannotspan and be anchored in the mammalian membrane, progeny virus cannot beproduced in mammalian cells. An advantage to the approach of the presentinvention is that the mutants are engineered as deletion mutants, hencethere is absolutely no chance for reversion to wild-type phenotype, acommon problem with virus vaccines.

[0039] The protocol described by the present invention works for anyvirus which replicates in insects and mammals and h as integral membraneproteins as part of its structure, namely, Togaviruses, Flaviviruses andBunya viruses and all other enveloped viruses which can replicatenaturally in both mammalian and insect cells, as well as envelopedviruses which can be made to replicate in mammalian and insect cells bygenetic engineering of either the virus or the cell.

[0040] Vaccines are made against any membrane-containing virus byremoving amino acids from the membrane-spanning domain of a protein inthe viral envelope. This is done by removing bases from a cDNA clone ofthe virus as described below. RNA transcribed from the altered clone istransfected into insect cells. The viruses produced are amplified byrepeated growth in insect cells until large quantities of mutant virusesare obtained. These viruses are tested for its ability to infect andproduce progeny in mammalian cells. Viruses which do not produce progenyin mammalian cells are tested for ability to produce immunity inlaboratory animals. Those viruses which do produce immunity arecandidates for production of human and animal vaccines by proceduresknown in the art.

[0041] Using the prototype of the Alphaviridea, Sindbis virus, thedifferent compositions of insect and mammalian membranes are exploitedto produce mutants which assemble efficiently in insect cells butassemble poorly in mammalian cells. The envelope glycoproteins ofSindbis virus are integrated into the membranes of the endoplasmicreticulum as a multi pass protein with 6 membrane spanning domains.There are, therefore, 6 potential targets for the production of deletionmutations which will prevent the correct integration of a transmembranedomain (TMD) (See FIG. 1). Some of these targets are less satisfactoryfor deletion mutagensis because they have functions other than simplyanchoring the protein in the membrane bilayer. For example,transmembrane domain #1 (FIG. 1) is the signal sequence which isrecognized by the Signal Recognition Particle and directs proteinsynthesis to the membranes of the ER. Truncating this domain wouldlikely disturb targeting in both mammalian and insect cells. TMD #3 willbecome a cytoplasmic domain upon protein maturation and containsspecific sequences that recognize and bind capsid protein. It has beenshown that this interaction is very specific in nature and requires thesequence that is in the transmembrane domain (Liu et al., 1996; Lopez etal., 1994). TMD #3, therefore, like TMD #1 has a functional as well as astructural component. A significant deletion in this domain would likelyeliminate budding in both cell systems. This leaves four transmembranedomains as targets for the production of deletions which will effectmembrane integration (FIG. 1, TMD #2, #4, #5, and #6).

[0042] The 6k protein is not a component of mature virus and itsfunction in virus assembly is not clear. In the poly protein the properintegration and orientation of 6k in the ER membrane is essential forthe correct integration of E1. The transmembrane domains of 6k (TMD #4and #5) are excellent targets for deletion mutation as failure tointegrate one of these domains may cause the poly protein to integrateinto the membrane in a wrong configuration or cause the failure tointegrate E1. Transmembrane domain #2 and #6 are the membrane spanningdomains of E2 and E1 and are both obvious targets for deletion mutation.Multiple membrane spanning domains in this poly protein suggest that ifdeletion mutations in a single transmembrane domain do not totally blockvirus production in mammalian cells, then deletions in additionalmembrane spanning domains can further reduce maturation to negligiblelevels.

[0043] The present invention is directed to a genetically engineeredmembrane-enveloped virus comprising a transmembrane protein which has adeletion of one or more amino acids in the transmembrane region of theprotein such that the transmembrane protein is able to span or correctlyintegrate into the membrane of an infected cell when the engineeredvirus replicates in insect cells, but is unable to span or integrateinto the membrane of an infected cell when the virus replicates inmammalian cells. Preferably, the virus is an Arthropod vectored virusselected from the group consisting of Togaviruses, Flaviviruses, Bunyaviruses and all other enveloped viruses which can replicate naturally inboth mammalian and insect cells, as well as enveloped viruses which canbe made to replicate in mammalian and insect cells by geneticengineering of either the virus or the cell. Representative examples ofsuch engineered viruses are ΔK391 virus, TM17 and TM16 virus. Stillpreferably, the insect cells are mosquito cells, such as Aedesalbopictus cells, and the mammalian cells are human cells.

[0044] In a preferred embodiment, the genetically engineered,membrane-enveloped virus is Sindbis virus, and the transmembrane proteinis viral glycoprotein E2. However, a person having ordinary skill inthis art could readily predict that similar mutations can b esuccessfully installed in the membrane spanning domains of other virusmembrane proteins such as E1.

[0045] In another preferred embodiment, the genetically engineeredmembrane-enveloped virus is selected from the group consisting of HSV,HIV, rabies virus, Hepatitis, and Respiratory Syncycial virus, and thetransmembrane protein is selected from the group consisting ofglycoprotein E1, glycoprotein E2, and G protein.

[0046] In still another preferred embodiment, the genetically engineeredmembrane-enveloped virus are RNA tumor viruses, and the transmembraneprotein is Env.

[0047] The present invention is also drawn to a method of producing aviral vaccine from the genetically engineered membrane-enveloped virusdisclosed herein for vaccination of mammals, comprising the steps ofintroducing the engineered virus into insect cells and allowing thevirus to replicate in the insect cells to produce a viral vaccine.Representative examples of the engineered viruses are ΔK391 virus, TM17virus and TM16 virus.

[0048] In addition, the present invention provides a method forvaccination of an individual in need of such treatment, comprising thesteps of introducing the viral vaccine of the present invention into theindividual and allowing the vaccine to produce viral proteins for immunesurveillance and stimulate immune system for antibody production in theindividual.

[0049] Furthermore, the present invention provides a method of producinga viral vaccine to a disease spread by a wild mosquito population to amammal, comprising the steps of genetically engineering a deletion ofone or more amino acids in a viral transmembrane protein to produce anengineered virus, wherein the transmembrane protein is able to span themembrane envelope when the virus replicates in mosquito cells, but isunable to span the membrane envelope when the virus replicates inmammalian cells, and wherein the virus remains capable of replicating inmosquito cells; introducing the engineered virus into a wild mosquitopopulation; and allowing the virus to replicate in cells of the wildmosquito population to produce a population of mosquitos which excludesthe wild type pathogenic virus and harbors the vaccine strain of thevirus such that the mosquito bite delivers the vaccine to a mammalbitten.

[0050] It is contemplated that pharmaceutical compositions may beprepared using the novel mutated viruses of the present invention. Insuch a case, the pharmaceutical composition comprises the novel virus ofthe present invention and a pharmaceutically acceptable carrier. Aperson having ordinary skill in this art readily would be able todetermine, without undue experimentation, the appropriate dosages androutes of administration of this viral vaccination compound. When usedin vivo for therapy, the vaccine of the present invention isadministered to the patient or an animal in therapeutically effectiveamounts, i.e., amounts that immunize the individual being treated fromthe disease associated with the particular virus. It will normally beadministered parenterally, preferably intravenously or subcutaineusly,but other routes of administration will be used as appropriate. Theamount of vaccine administered will typically be in the range of about10³ to about 10⁶ pfu/kg of patient weight. The schedule will becontinued to optimize effectiveness while balancing negative effects oftreatment. See Remington's Pharmaceutical Science, 17th Ed. (1990) MarkPublishing Co., Easton, Penn.; and Goodman and Gilman's: ThePharmacological Basis of Therapeutics 8th Ed (1990) Pergamon Press;which are incorporated herein by reference. For parenteraladministration, the vaccine will be most typically formulated in a unitdosage injectable form (solution, suspension, emulsion) in associationwith a pharmaceutically acceptable parenteral vehicle. Such vehicles arepreferably non-toxic and non-therapeutic. Examples of such vehicles arewater, saline, Ringer's solution, dextrose solution, and 5% human serumalbumin.

[0051] The following examples are given for the purpose of illustratingvarious embodiments of the invention and are not meant to limit thepresent invention in any fashion:

EXAMPLE 1

[0052] A Single Amino Acid Deletion Mutant, K391

[0053] Using the full length clone of the Alpha virus Sindbis describedpreviously (Liu et al 1996, Rice et al., 1987), a deletion removing 3bases encoding a lysine at position 391 in the amino acid sequence ofthe virus glycoprotein E2 has been constructed. This lysine is part ofthe putative membrane-spanning domain of this protein (Rice et al 1982).

[0054] Site-directed mutagenesis was used to generate a deletion mutant(Lys391) in Toto 1101, a plasmid containing the full-length Sindbis cDNAand an SP6 promoter that can be used to transcribe infectious RNA fromthe clone in vitro (Rice et al., 1987; Liu and Brown, 1993a). Using themegaprimer method of PCR mutagenesis (Sarkar and Sommer, 1990) describedpreviously (Liu and Brown, 1993a), three nucleotides (nucleotides 9801,9802, 9803) were removed in the cDNA clone of Toto 1101, resulting inthe removal of the codon AAA (K391).

[0055] A 30 base oligonucleotide of the sequence,5′CTCACGGCGCGCACAGGCACATAACACTGC3′ (SEQ ID No.: 1) was used as themutagenesis primer. This primer, along with the “forward primer”5′CCATCAAGCAGTGCGTCG3′ (SEQ ID No.: 2; 18 mer), generated a 518 base“Megaprimer” (nucleotides (nts) 9295-9813). The second PCR reactionconsisted of 0.5 μg of megaprimer, 100 μg Toto 1101 template and 0.5 μgof the “reverse primer” 5′ GGCAGTGTCCACCTTAATCGCCTGC 3′ (SEQ ID No.: 3).All PCR reactions employed 30 cycles at 95 degrees for 1 min., 64degrees for 1 min., 72 degrees for 1 min. and a final incubation at 72degrees for 8 min. The resulting PCR product (1149 nts) was cleaved withBCL I and SPL and inserted into the corresponding site in Toto 1101,creating the deletion mutant K391. After the deletion was confirmed bydideoxynucleotide sequencing through the entire subcloned region usingSequenase™ (U.S. Biochemical, Cleveland, Ohio.), infectious RNA wastranscribed in vitro using SP6 polymerase and was introduced into BHK-21cells.

EXAMPLE 2

[0056] In Vitro Transcription and RNA Transfection Of K391

[0057] Plasmid DNA containing the full-length cDNA copy of Sindbis virusK391 or wild type RNA was linearized with XhoI and transcribed in vitrowith SP6 RNA polymerase as described previously (Rice et. al., 1987). 1μg of Xho I linearized K391 cDNA or wild type Sindbis virus cDNA wastranscribed in buffer consisting of 80 mM Hepes, pH 7.5, 12 mM MgCl, 10mM DTT, 2 mM spermidine and 100 μgm BSA with 3 mM each ATP, UTP, CTP,1.5 mM GTP and 4.5 mM m⁷ GpppG, 20 units SP6 RNA polymerase and 20 unitsRNase inhibitor in a 20 μl reaction volume. After incubation at 37° C.for 2 hours, RNA production was assayed by running 2 μl of the RNAproduct on a 1% agarose gel.

[0058] Baby Hamster Kidney (BHK21) cells and Aedes albopictus (mosquito)cells were transfected with RNA derived from the mutant or wild typeclone. Mosquito cell transfections were carried out using 5×10⁶ cellsresuspended in RNase free electroporation buffer consisting of 20 mMHepes pH 7.05, 137 mM NaCl, 0.7 mM Na₂HPO₄ and 6 mM dextran. Washedcells were resuspended in diethyl pyrocarbonate (DEPC) treated water toa concentration of 5×10⁷ cells/ml. RNA transcripts in 20 μl were addedto 400 μl washed cells and transferred to a 0.2 cm gap length cuvette.Optimal electroporation parameters for these cells was found to be 2 KV25 μF, 8 resistance. Transfected cells were incubated at 37° C. untilcytopathic effect was observed (about 24 hours).

[0059] After 24 hours of incubation, the media was collected from bothinfected cell lines as well as non-RNA transfected controls. The mediafrom each cell line was tested for the presence of infectious virus byplaque assay (as described by Renz and Brown 1976) on mosquito andBHK-21 cell monolayers (Table 1). TABLE 1 Infectious virus produced bytransfection of BHK21 or Aedes albopictus (AA) cells with Sindbis viruswild type (wt) or mutant K391 Cell BHK BHK AA AA line Mock^(a) with Mockwith Trans- Trans- BHK with K391 Trans- AA with K391 fected fected wtRNA RNA fected wt RNA RNA Media no virus 1.5 × 10⁹ 3.0 × 10³ no virus5.0 × 10⁸ 1.0 × titered detected virus/ml detected virus/ml 10⁸ on BHKMedia no virus 8 × 10⁷ 8.0 × 10⁴ no virus 1.0 × 10⁹ 2.0 × titereddetected virus/ml detected virus/ml 10⁹ on AA virus/ml

[0060] As shown in Table 1, the mutant K391 produces significant amountsof infectious virus particles only when replicating in the insect cell.BHK cells transfected with K39 1 produced very low levels of virus, 4 to5 orders of magnitude lower than the amount produced in insect cells.

EXAMPLE 3

[0061] Metabolic Radioactive Labeling of Viral Proteins

[0062] Subconfluent monolayers of BHK21 cells in 25 cm² flasks weretransfected with wild type or K391 mutant RNA as described above.Monolayers were starved for 30 min in methionine- and cysteine-freemedium (MEM-E) containing 1% FCS, 2 mM glutamine and 5% TPB (starvationmedium). At 16 hours post-transfection, cells were pulse-labeled withstarvation medium containing 50 μCi/ml [³⁵S] Met/Cys protein labelingmix for 20 minutes. Labeling was terminated by washing the monolayerswith PBS containing 75 μg/ml cycloheximide. Monolayers were chased for45 minutes in medium containing 10 times the normal concentration ofmethionine and cysteine and 75 μg/ml cycloheximide.

EXAMPLE 4

[0063] Immunoprecipitation and Polyacrylamide Gel Electrophoresis

[0064] Radiolabeled viral proteins were immunoprecipitated with antiseraas described (Knipfer and Brown, 1989). [³⁵S] Met/Cys labeled cells werewashed twice in cold PBS and lysed in lysis buffer: 0.5% NP-40, 0.02 MTris HCl pH 7.4, 0.05 M NaCl, 0.2 mM PMSF, 0.2 mM TPCK and 0.02 mM TLCK.The nuclei were pelleted by centrifugation and discarded. Thesupernatant was pre-absorbed with 100 μl of protein A/Sepharose beads(Sigma) suspended in lysis buffer for 1 hr, and the beads were pelleted.The pre-absorbed supernatant was treated with 200 μl of proteinA/Sepharose beads coupled to rabbit anti-SVHR serum or E2 tailmonospecific polyclonal serum and agitated overnight at 4° C. Theimmunoprecipitated bead-antibody-protein complexes were washed threetimes with lysis buffer and then solubilized in SDS-PAGE sample bufferconsisting of 12% glycerol, 4% SDS, 50 mM Tris pH 6.8, 5%mercaptoethanol and 0.02% bromphenol blue. The samples were heated for 3min at 95° C. and the beads were removed from the sample bycentrifugation. Gel electrophoresis was carried out on a 10.8% SDS-PAGEor 16% Tricine gel as described previously (Liu and Brown, 1993 a,b).Fluorography was performed as described (Bonner and Laskey, 1974) anddried gels were exposed to Kodak XAR-5 film (see FIG. 2).

EXAMPLE 5

[0065] Transmission Electron Microscopy

[0066] BHK-21 cell monolayers infected with K391 produced fromtransfected mosquito cells or transfected with K391 RNA were lifted fromflasks by trypsin treatment at desired time points, and the cells werepelleted by low speed centrifugation. Cell pellets were washed twice inPBS and fixed in 4% glutaraldehyde at 4° C. overnight. The cells werethen washed three times with 0.2 M cacodylate buffer (pH 7.2),post-fixed with 2% osmium tetroxide for 1 hour at room temperature, andwashed three times in cacodylate buffer. The cells were stained en blocfor 1 hr at room temperature with 0.5% uranyl acetate. After threewashes, cell pellets were embedded in 1% agarose and dehydrated througha graded ethanol/acetone series. Final embedding was in Mollenhauer's(1964) Epon-Araldite epoxy mixture #1 at 70° C. for two days. Ultrathinsections were cut on a Sorvall MT5000 microtome and collected on 150mesh copper grids. Sections were stained with 1% uranyl acetate and/orlead citrate and were photographed in a Jeol 100CX transmission electronmicroscope (see FIG. 3).

[0067] Although BHK cells infected with K391 virus or transfected withK391 RNA produce no virus detectable by the plaque assay, it was shownby PAGE that they do produce all virus structural proteins (FIG. 2).Further, it was shown by electron microscopy that they assemble theintracellular (non infectious) virus cores (FIG. 3).

[0068] Delta K391 produces very high titers of mutant Sindbis virusparticles when allowed to replicate in mosquito cells. The exposedregions of the proteins (ecto domains) are wild type in sequence. Thesewild type proteins allow the virus to enter mammalian cells and producevirus proteins (see FIG. 2) but new virus is not assembled as shown byelectron microscopy in FIG. 3.

[0069] Delta K391 is a vaccine strain. It is produced in very highconcentration in cultured insect cells. When the virus is injected intoa mammalian host, the virus circulates and infects cells in themammalian host. These infected cells produce and present virus proteinsfor immune surveillance. However, the infection will be limitedprimarily to those cells infected initially by the innoculum because ofthe truncation in the membrane domain of the viral glycoprotein. Becausethe vaccine strain is the result of a deletion mutation, reversion towild type pathogenic phenotype is not possible.

[0070] Furthermore, an engineered deletion mutant may be introduced intothe wild mosquito population. It has been shown that these viruses arespread from the female parent to the progeny by a process oftransovariol transmission (Leakey 1984). When these mosquitoes bite avertebrate they will provide an immunizing dose (10⁶ infectiousparticles) of the vaccine strain (for example, Delta K391). Karpf et al(1997) showed that infection of insect cells by one Alpha virus preventsthe cells from being infected by another, even distantly-related alphavirus for an indefinite amount of time (over two years in cell culture,where the life of a mosquito is 28 days). Thus, the presence of thevaccine strain such as K391 or other deletion mutants described in thepresent invention will block the spread of other related and pathogenicviruses by these insects

EXAMPLE 6

[0071] Deletion In The E2 Transmembrane Domain

[0072] Protocols for testing the requirements placed on thetransmembrane domain of E2 (FIG. 1, TMD #2) is given. This protocol canbe easily replicated for any other of the Sindbis membrane spanningdomains or the membrane spanning domains of any other virusglycoprotein. The hydrophobic Sindbis PE2 membrane anchor consists of 26amino acids. As is common with other membrane spanning domains littleamino acid homology is conserved among the alphaviruses, although thelength of this hydrophobic region is highly conserved (Strauss andStrauss, 1994). The lack of sequence conservation in this domainsuggests that it is the hydrophobic properties of the domain and not itssequence which is critical for integration.

[0073] The transmembrane domain of E2 begins at amino acid 365 of thePE2 sequence. This hydrophobic region consists of the sequence:VYTILAVASATVAMMIGVTVAVLCAC (SEQ ID No.: 4). Adams and Rose (1985)demonstrated that a minimum of 14 amino acids in the transmembranedomain of the VSV G protein were necessary for proper anchoring inmammalian cells. Therefore, mutagenic primers have been designed whichcreate a nested set of deletions in the E2 transmembrane domain.Beginning with a deletion of 16 amino acids (which leaves 10 amino acidsin the hydrophobic region), a set of deletions were constructed whichdelete from as many as 16 amino acids, to as few as 1 amino acid fromthe membrane anchor (FIG. 4).

[0074] Deletions were constructed using PCR megaprimer mutagenesis togenerate deleted fragments containing unique BclI and SplI sites. Allresulting constructs were installed into the wild-type Sindbis cDNAconstruct Toto Y420 to generate the mutant plasmids. After linearizationwith XhoI and transcription using SP6 polymerase, transcripts weretransfected into BHK or Aedes albopictus cells by electroporation asdescribed above. Production of infectious virus from these transfectionswere titered on both BHK and C710 mosquito cells to determine the hostrange of these constructs. Table 2 shows the deleted sequences and theprimer sequences used in their construction.

[0075] For each construct the same primer pair is used to generate theentire BclI to SplI region. The forward primer E1Bcl21 is comprised ofthe sequence from nucleotide 9306-9327 and reads from 5′-3′GCGTCGCCFATAAGAGCGACC (SEQ ID No.: 5). The reverse primer Splext iscomprised of the sequence from nucleotide 10420-10444 which is thecomplementary sequence reading from 5′-3′ CAGTGTGCACCTTAATCGCCTGC (SEQID No.: 6).

[0076] The virus produced by transfection of insect cells is tested forits ability to produce plaques in BHK and C7-10 mosquito cells as forthe mutant E2 ΔK391. Those mutants which do not produce plaques in BHKcells are tested for their ability to infect BHK cell relative to wildtype virus by immunofluorescence assay of infected monolayers. Thislater assay is compared to the total protein in purified preparations ofthe mutant and wild type virus to establish the relative infectivity ofeach mutant population. The goal is to truncate the transmembrane domainas much as possible and still obtain reasonable amounts of virus inC7-10 mosquito cell monolayers which can infect but not produce maturevirus in BHK cells. Additional transmembrane domains (up to fourdomains) can be truncated in circumstances where truncation of a singletransmembrane domain reduces but does not eliminate virus growth in BHKcells.

[0077] The length of the transmembrane (TM) domain of E2 wassystematically reduced from 26 amino acids to 10, 12, 14, 16, 17 and 18amino acids, and the effects of these truncations on the ability ofthese viruses to replicate in cells of the vertebrate (BHK-21, hamstercells) and invertebrate (Aedes albopictus, mosquito cells) hosts wereexamined. Table 3 presents results typical of several of suchexperiments. The data reveal that reducing the transmembrane domain from26 to 10 amino acids or 12 amino acids results in viruses incapable ofefficient assembly in either host. Increasing the length of thetransmembrane domain to 14 amino acids results in viruses that growpoorly in mammalian cells but somewhat better in insect cells.Increasing transmembrane domain length to 16 or 17 amino acids restoreswild type levels of growth in insect cells while growth in mammaliancells remains greatly impaired. Increasing the length of thetransmembrane domain to 18 amino acids restores growth in mammaliancells. The reduction in the length of the transmembrane domain of the E2glycoprotein has resulted in the production of virus mutants in whichefficient growth is restricted to insect cells. The accepted terminologyfor such mutations is “host range mutation”.

[0078] The data presented above show that large deletions in thetransmembrane domains of the glycoproteins of insect vectored virusescan result in the restriction of virus assembly to insect cells. Mutantswhich produce low levels of virus (transmembrane domains 10, 12, 14) areunable to correctly integrate the membrane proteins into the host cellmembranes. The less impaired mutants, represented by transmembranedomain 16 and transmembrane domain 17, can infect mammalian cells,produce structural proteins, and form nucleocapsid structures containingthe viral RNA. However, these mutants are defective in steps in virusassembly. TABLE 2 Listing of the deletions in Sindbis E2 and the primersused Primer-Designated by No. of Transmembranal NucleotidesOligonucleotide Sequence of Amino Acids Deleted Mutagenic Primer(Negative Strand) E2 TM10 9734-9782 ACATAACACTGCGATGGTGTACAC (SEQ IDNo.: 7) E2 TM12 9740-9782 ACATAACACTGCGGCTAAGATGG (SEQ ID No.: 8) E2TM14 9746-9782 ACATAACACTGCTGCGACGGCT (SEQ ID No.: 9) E2 TM16 9743-9773GCAACAGTTACGACGGCTAAG (SEQ ID No.: 10) E2 TM17 9743-9770ACAGTTACGCCGACGGCTAAG (SEQ ID No.: 11) E2 TM18 9743-9767GTTACGCCAATGACGGCTAAG (SEQ ID No.: 12) E2 TM19 9743-9764CGCCAATCATGACGGCTAAGA (SEQ ID No.: 13) E2 TM20 9755-9773GCAACAGTTACGGTAGCTGA (SEQ ID No.: 14) E2 TM21 9755-9770AGTTACGCCGGTAGCTGA (SEQ ID No.: 15) E2 TM22 9761-9773TGCAACAGTTACCGCCACGGT (SEQ ID No.: 16) E2 TM23 9761-9770ACAGTTACGCCCGCCACGGT (SEQ ID No.: 17) E2 TM24 9761-9767GTTACGCCAATCGCCACGGT (SEQ ID No.: 18) E2 TM25 9761-9764ACGCCAATCATCGCCACGGT (SEQ ID No.: 19)

[0079] TABLE 3 Growth of Sindbis virus TM deletion mutants in insect andvertebrate cells Growth in insect Growth in mammalian Mutant^(°) cells(pfu/ml)− cells (pfu/ml)− Wild type 5 × 10⁹ 5 × 10⁹ TM 10 2 × 10³ 3 ×10⁴ TM 12 5 × 10³ 6 × 10² TM 14 6 × 10⁷ 4 × 10² TM 16 2 × 10⁹ 7 × 10⁴ TM17 3 × 10⁹ 1 × 10⁵ TM 18 1 × 10⁸ 6 × 10⁸

[0080] Mutants were constructed using the Stratagene Quick change®mutagenesis protocol using a cDNA template containing the virusstructural genes. Desired mutations were subcloned into the full lengthvirus cDNA vector containing an SP6 promoter for the transcription offull length infectious viral RNA. Mutant transcripts were transfectedinto—mosquito cells or—mammalian BHK cells and incubated for theappropriate time and temperature before harvesting the virus-containingmedia. Virus yields from both cell types were assayed by titration onmonolayers of BHK cells.

EXAMPLE 7

[0081] Uses of Deletion Mutants As Vaccine

[0082] Mutations which restrict the assembly of virions only to insectcells suggest that viruses produced from these cells may be used toinfect an animal which could only produce low numbers of progenyviruses. Such a phenotype could result in the production of protectiveimmunity in that animal without pathological Id consequences. Mutants TM16 and 17 were selected for further study to determine their potentialfor producing protective immunity. The results of these experiments arepresented in Table 4. TABLE 4 Protection Of Adult Mice From SindbisVirus By Vaccination With TM Mutations Morbitity Mortality MOR- MOR-POST POST VIRUS DOSE TALITY BIDITY CHALLENGE CHALLANGE CHALLENGE Mock10⁶ 0% 0% SAAR86 1000 92% 36% (buffer) pfu i.c. TM16 10⁶ 0% 0% SAAR861000 68% 48% pfu i.c. TM16 NA 0% 0% SAAR86 1000 92% 36% UV pfu i.c. TM1710⁶ 0% 0% SAAR86 1000 0 0 pfu i.c. TM17 NA 0% 0% SAAR86 1000 84% 36% UVpfu i.c.

[0083] Twenty five 21 days old CD-1 mice were used in each study. Mutantviruses from transfected Aedes albopictus U4.4 cells was injected intothe mice subcutaneously at the dose indicated. Fourteen days after theinitial injection the mice were challenged with the SAAR 86 strain ofSindbis virus as indicated.

[0084] TM 16 was a poor vaccine compared to TM 17 although bothmutations showed identical phenotypes in the tissue culture cell systemdescribed in Table 3. It is clear that the protection achieved byinjection with TM 17 was not the simple result of exposure to virusprotein, as the UV treated virus did not protect.

[0085] To further elucidate the mouse response to these two mutants,serum of vaccinated mice was tested for the presence of circulatingantibody by standard ELISA assay. The results of this experiment isshown in FIG. 5.

[0086] Mutants TM16 and TM 17 appeared to produce similar levels ofcirculating antibody as would be expected from an inoculation with thesame quantity of virus. This result suggested that antibodies capable ofbinding to denatured virus, as well as infectious virus, were induced inthe mice at roughly equivalent levels by both mutants.

[0087] The results presented in FIG. 5 suggested that the immuneresponse to TM 17 was different from the response to TM16. The serum ofthe vaccinated mice was therefore examined for the presence ofneutralizing antibody to Sindbis virus. The results are shown in FIG. 6.By contrast with mutant TM16, mutant TM 17 induced significantly moreneutralizing antibody. This likely explains its superior performance asa vaccine.

[0088] The transmembrane domains of the glycoproteins of Alpha, Flaviand Bunya viruses which have been sequenced reveal that they have thecommon property of being hydrophobic sequences that are predicted toform alpha helices in membrane bilayers. It is predicted that truncationof the transmembrane domain described above for an alphavirus willproduce a similar pattern of host restriction in any one of theseviruses. Thus, the protocol described above has the potential ofproducing live vaccines against any one of these agents. Because themutations are large deletions, there is little prospect of spontaneousreversion to wild type virus. Indeed, in the time these mutants wereexamined in the laboratory, no such revertants have been detected.

[0089] The observation that TM16 and 17 have such different propertiesin terms of their ability to produce protection as a vaccine whilehaving similar growth characteristics in cell culture is mostinteresting. The data showing that TM 17 produces a higher level ofneutralizing antibody suggests that TM 17 may be structurally moreidentical to wild type virus than TM 16. A possible explanation for thismay lie in the very precise structure of the virion itself. The surfaceof Sindbis is a T=4 icosahedral shell made rigid by scaffoldinginteraction among the E1 glycoproteins. In the mature virion, the E1glycoprotein is a highly constrained energy-rich metastable structure.The energy stored in E1 is believed to be used to disassemble theprotein lattice and to allow virus-cell membrane fusion. The constrainedform of E1 is developed in the endoplasmic reticulum of infected cellsby folding through several disulfide bridged intermediates as the PE2-E1heterotrimer is produced. The energy rich form of E1 rapidly reorganizesto a lower energy state by the reshuffling of disulfide bridges if theprotein is isolated from the virion in the absence of thiol blockingagents. It has also been demonstrated that the function of the membraneglycoprotein is affected by mutations in the core protein, suggestingthat specific interactions between the capsid and the membrane proteinE2 are critical to virus stability. The rigid organization of the virusmembrane glycoproteins and the identical structure of the inner core mayrequire the E2 endodomain (cytoplasmic location), which binds to thevirus core via interactions with the capsid protein hydrophobic pocket,emerges from the membrane in a particular orientation. The correctorientation may be required for the very specific binding of the E2endodomain to the hydrophobic cleft in the capsid protein. As aminoacids are removed from the transmembrane domain helix, the orientationof the E2 tail may be altered at the point of egress from the membranebilayer.

[0090] Alternatively, deletions in the transmembrane domain of SindbisE2 may distort the E2 ectodomain, the domain oriented toward theexterior of the cell, thereby destabilizing interactions with thescaffolding protein E1. In the case of TM 16, this may result in anassociation that allows for virus assembly but which produces arelatively unstable virion. This instability may result in thespontaneous reshuffling of disulfide bridges in the E1 glycoprotein to alow energy, non-native state which is antigenically dissimilar fromnative protein and may cause the structural degradation of the virion.The E2 tail of the mutant TM 17 contains an additional amino acid in thetransmembrane domain helix and is predicted to exit the membrane at aposition 100° distant to that of the TM 16 mutant. This may relievesufficient structural strain to allow the mutant to remain stable andimmunogenic.

[0091] In summary, differences in the structure and physical propertiesof insect and mammalian cells have been exploited to produce host rangemutations with potential as vaccines. This approach should be applicableto the production of vaccines against any of the several hundredmembrane-containing insect borne viruses for which a cDNA clone can beproduced.

[0092] One skilled in the art will appreciate readily that the presentinvention is well adapted to carry out the objects and obtain the endsand advantages mentioned, as well as those objects, ends and advantagesinherent herein. The present examples, along with the methods,procedures, treatments, molecules, and specific compounds describedherein are presently representative of preferred embodiments, areexemplary, and are not intended as limitations on the scope of theinvention. Changes therein and other uses will occur to those skilled inthe art which are encompassed within the spirit of the invention asdefined by the scope of the claims.

[0093] The following references were cited herein:

[0094] Adams and Rose. (1985) Cell 41(3):1007-15.

[0095] Berge (ed.) (1975): International Catalogue of Arboviruses; 2nded., DHEW Publ. No. (CDC) 75-8301 (U.S. Government Office, Washington,D.C.)

[0096] Bonner and Laskey. (1974). Eur. J. Biochem. 46:83-88.

[0097] Bowers et al. (1995). Virology 212: 1-12.

[0098] Brown and Condreay (1986). Replication of alphaviruses inmosquito cells. In The Togaviridae and Flaviviridae. S. Schlesinger(ed.), pp. 473-501.

[0099] Clayton. (1964) J. Lipid Res. 5:3-19.

[0100] Karpf et al. (1997) J. Virol.71:7119.

[0101] Knipfer and Brown. (1989). Virology 170:117-122.

[0102] Leake. (1984). Transovarial transmission of arboviruses bymosquitoes. In Vectors in Virus Biology (Mayo and Harrap, eds.), pp.63-92. Academic Press.

[0103] Liu and Brown (1993a). J. Cell Biol. 120:877-883.

[0104] Liu and Brown (1993b). J. Virol., 196:703-711.

[0105] Liu et al. (1996) Virology 222: 236-246.

[0106] Mitsuhashi et al. (1983). Cell Biol. Int. Rep. 7:1057-1062.

[0107] Mollenhauer. (1964). Stain Techn. 39:111-114.

[0108] NIAID Report of the Task Force on Microbiology and InfectiousDiseases (1992). NIH Publication No. 92-3320.

[0109] Renz and Brown. (1976). J. Virol. 19:775-781.

[0110] Rice et al. (1982). J. Mol. Biol. 154:355-378.

[0111] Rice et al. (1987). J. Virol. 61:3809-3819.

[0112] Sarkar and Sommer. (1990). BioTechniques. 8:404-407.

[0113] Schlesinger and Schlesinger (1990). “Replication of Togaviridaeand Flaviviridae.” (D. M. Knipe and B. N. Fields, eds.), In VirologyVol. I, pp. 697-711. Raven Press, Ltd., New York.

[0114] Sprenger and Wuithiranyagool (1985). J. Am. Mosquito ControlAssoc. 2:217-219.

[0115] Any patents or publications mentioned in this specification areindicative of the levels of those skilled in the art to which theinvention pertains. Further, these patents and publications areincorporated by reference herein to the same extent as if eachindividual publication was specifically and individually indicated to beincorporated by reference.

1 34 1 30 DNA artificial sequence Used as the mutagenesis primer withthe “forward primer” to generate a 518 base “Megaprimer” correspondingto nucleotides 9295-9813. 1 ctcacggcgc gcacaggcac ataacactgc 30 2 18 DNAartificial sequence Used as the “forward primer” with the mutagenesisprimer to generate a 518 base megaprimer corresponding to nucleotides9295-9813. 2 ccatcaagca gtgcgtcg 18 3 25 DNA artificial sequence Used asthe “reverse primer” with the megaprimer and the Toto 1101 plasmidtemplate to create 1149 nucleotide product used to create the deletionmutant K391 in Toto 1101. 3 ggcagtgtgc accttaatcg cctgc 25 4 26 PRTSindbis virus transmembrane domain of E2 in the PE2 sequence 365..390 4Val Tyr Thr Ile Leu Ala Val Ala Ser Ala Thr Val Ala Met Met 5 10 15 IleGly Val Thr Val Ala Val Leu Cys Ala Cys 20 25 5 21 DNA artificialsequence 9306-9327 Forward primer E1Bcl21 from megaprimer used withreverse primer to generate deletion constructs containing unique BclIand SplI sites. 5 gcgtcgccta taagagcgac c 21 6 23 DNA artificialsequence 10420-10444 Reverse primer Splext from megaprimer used withforward primer to generate deletion constructs containing unique BclIand SplI sites. 6 cagtgtgcac cttaatcgcc tgc 23 7 24 DNA artificialsequence Mutagenic primer E2 TM10 (negative strand) used to create adeletion in the E2 transmembranal domain in the Sindbis viralglycoprotein. 7 acataacact gcgatggtgt acac 24 8 23 DNA artificialsequence Mutagenic primer E2 TM12 (negative strand) used to create adeletion in the E2 transmembranal domain in the Sindbis viralglycoprotein. 8 acataacact gcggctaaga tgg 23 9 22 DNA artificialsequence Mutagenic primer E2 TM14 (negative strand) used to create adeletion in the E2 transmembranal domain in the Sindbis viralglycoprotein. 9 acataacact gctgcgacgg ct 22 10 21 DNA artificialsequence Mutagenic primer E2 TM16 (negative strand) used to create adeletion in the E2 transmembranal domain in the Sindbis viralglycoprotein. 10 gcaacagtta cgacggctaa g 21 11 21 DNA artificialsequence Mutagenic primer E2 TM17 (negative strand) used to create adeletion in the E2 transmembranal domain in the Sindbis viralglycoprotein. 11 acagttacgc cgacggctaa g 21 12 21 DNA artificialsequence Mutagenic primer E2 TM18 (negative strand) used to create adeletion in the E2 transmembranal domain in the Sindbis viralglycoprotein. 12 gttacgccaa tgacggctaa g 21 13 21 DNA artificialsequence Mutagenic primer E2 TM19 (negative strand) used to create adeletion in the E2 transmembranal domain in the Sindbis viralglycoprotein. 13 cgccaatcat gacggctaag a 21 14 20 DNA artificialsequence Mutagenic primer E2 TM20 (negative strand) used to create adeletion in the E2 transmembranal domain in the Sindbis viralglycoprotein. 14 gcaacagtta cggtagctga 20 15 18 DNA artificial sequenceMutagenic primer E2 TM21 (negative strand) used to create a deletion inthe E2 transmembranal domain in the Sindbis viral glycoprotein. 15agttacgccg gtagctga 18 16 21 DNA artificial sequence Mutagenic primer E2TM22 (negative strand) used to create a deletion in the E2transmembranal domain in the Sindbis viral glycoprotein. 16 tgcaacagttaccgccacgg t 21 17 20 DNA artificial sequence Mutagenic primer E2 TM23(negative strand) used to create a deletion in the E2 transmembranaldomain in the Sindbis viral glycoprotein. 17 acagttacgc ccgccacggt 20 1820 DNA artificial sequence Mutagenic primer E2 TM24 (negative strand)used to create a deletion in the E2 transmembranal domain in the Sindbisviral glycoprotein. 18 gttacgccaa tcgccacggt 20 19 20 DNA artificialsequence Mutagenic primer E2 TM25 (negative strand) used to create adeletion in the E2 transmembranal domain in the Sindbis viralglycoprotein. 19 acgccaatca tcgccacggt 20 20 84 DNA Sindbis virusnucleotide sequence of the E2 transmembranal domain of the Sindbis viralglycoprotein 9717..9800 20 catcctgtgt acaccatctt agccgtcgca tcagctaccgtggcgatgat 50 gattggcgta actgttgcag tgttatgtgc ctgt 84 21 28 PRT Sindbisvirus amino acid sequence of the E2 transmembranal domain of the Sindbisviral glycoprotein 363..390 21 His Pro Val Tyr Thr Ile Leu Ala Val AlaSer Ala Thr Val Ala 5 10 15 Met Met Ile Gly Val Thr Val Ala Val Leu CysAla Cys 20 25 22 27 PRT artificial sequence Sequence of the E2transmembranal domain of the Sindbis viral glycoprotein after deletingamino acid 378, the resulting deletion mutant is TM25. 22 His Pro ValTyr Thr Ile Leu Ala Val Ala Ser Ala Thr Val Ala 5 10 15 Met Ile Gly ValThr Val Ala Val Leu Cys Ala Cys 20 25 23 26 PRT artificial sequenceSequence of the E2 transmembranal domain of the Sindbis viralglycoprotein after deleting amino acids 378 and 379, the resultingdeletion mutant is TM24 23 His Pro Val Tyr Thr Ile Leu Ala Val Ala SerAla Thr Val Ala 5 10 15 Ile Gly Val Thr Val Ala Val Leu Cys Ala Cys 2025 24 25 PRT artificial sequence Sequence of the E2 transmembranaldomain of the Sindbis viral glycoprotein after deleting amino acids 378through 380, the resulting deletion mutant is TM23 24 His Pro Val TyrThr Ile Leu Ala Val Ala Ser Ala Thr Val Ala 5 10 15 Gly Val Thr Val AlaVal Leu Cys Ala Cys 20 25 25 24 PRT artificial sequence Sequence of theE2 transmembranal domain of the Sindbis viral glycoprotein afterdeleting amino acids 378 through 381, the resulting deletion mutant isTM22. 25 His Pro Val Tyr Thr Ile Leu Ala Val Ala Ser Ala Thr Val Ala 510 15 Val Thr Val Ala Val Leu Cys Ala Cys 20 26 23 PRT artificialsequence Sequence of the E2 transmembranal domain of the Sindbis viralglycoprotein after deleting amino acids 376 through 380, the resultingdeletion mutant is TM21. 26 His Pro Val Tyr Thr Ile Leu Ala Val Ala SerAla Thr Gly Val 5 10 15 Thr Val Ala Val Leu Cys Ala Cys 20 27 22 PRTartificial sequence Sequence of the E2 transmembranal domain of theSindbis viral glycoprotein after deleting amino acids 376 through 381,the resulting deletion mutant is TM20. 27 His Pro Val Tyr Thr Ile LeuAla Val Ala Ser Ala Thr Val Thr 5 10 15 Val Ala Val Leu Cys Ala Cys 2028 21 PRT artificial sequence Sequence of the E2 transmembranal domainof the Sindbis viral glycoprotein after deleting amino acids 372 through378, the resulting deletion mutant is TM19. 28 His Pro Val Tyr Thr IleLeu Ala Val Met Ile Gly Val Thr Val 5 10 15 Ala Val Leu Cys Ala Cys 2029 20 PRT artificial sequence Sequence of the E2 transmembranal domainof the Sindbis viral glycoprotein after deleting amino acids 372 through379, the resulting deletion mutant is TM18. 29 His Pro Val Tyr Thr IleLeu Ala Val Ile Gly Val Thr Val Ala 5 10 15 Val Leu Cys Ala Cys 20 30 19PRT artificial sequence Sequence of the E2 transmembranal domain of theSindbis viral glycoprotein after deleting amino acids 372 through 380,the resulting deletion mutant is TM17. 30 His Pro Val Tyr Thr Ile LeuAla Val Gly Val Thr Val Ala Val 5 10 15 Leu Cys Ala Cys 31 18 PRTartificial sequence Sequence of the E2 transmembranal domain of theSindbis viral glycoprotein after deleting amino acids 372 through 381,the resulting deletion mutant is TM16. 31 His Pro Val Tyr Thr Ile LeuAla Val Val Thr Val Ala Val Leu 5 10 15 Cys Ala Cys 32 16 PRT artificialsequence Sequence of the E2 transmembranal domain of the Sindbis viralglycoprotein after deleting amino acids 373 through 384, the resultingdeletion mutant is TM14. 32 His Pro Val Tyr Thr Ile Leu Ala Val Ala AlaVal Leu Cys Ala 5 10 15 Cys 33 14 PRT artificial sequence Sequence ofthe E2 transmembranal domain of the Sindbis viral glycoprotein afterdeleting amino acids 371 through 384, the resulting deletion mutant isTM12. 33 His Pro Val Tyr Thr Ile Leu Ala Ala Val Leu Cys Ala Cys 5 10 3412 PRT artificial sequence Sequence of the E2 transmembranal domain ofthe Sindbis viral glycoprotein after deleting amino acids 369 through384, the resulting deletion mutant is TM10. 34 His Pro Val Tyr Thr IleAla Val Leu Cys Ala Cys 5 10

What is claimed is:
 1. A genetically engineered membrane-enveloped viruscomprising a viral transmembrane glycoprotein that is able to span orcorrectly integrate into the membrane of insect cells but not that ofmammalian cells due to deletion of one or more amino acids in thetransmembrane regions of said viral transmembrane glycoprotein, whereinsaid virus is capable of infecting and producing progeny virus in insectcells, and is capable of infecting but not producing progeny virus inmammalian cells.
 2. The genetically engineered membrane-enveloped virusof claim 1, wherein said virus is an Arthropod vectored virus.
 3. Thegenetically engineered membrane-enveloped virus of claim 1, wherein saidvirus is selected from the group consisting of Togaviruses,Flaviviruses, Bunya viruses, enveloped viruses capable of replicatingnaturally in mammalian and insect cells, and enveloped viruses whichreplicate in mammalian and insect cells as a result of geneticengineering of either the virus or the cell.
 4. The geneticallyengineered membrane-enveloped virus of claim 1, wherein said insectcells are mosquito cells.
 5. The genetically engineeredmembrane-enveloped virus of claim 4, wherein said mosquito cells areAedes albopictus cells.
 6. The genetically engineered membrane-envelopedvirus of claim 1, wherein said mammalian cells are human cells.
 7. Thegenetically engineered membrane-enveloped virus of claim 1, wherein saidvirus is selected from the group consisting of HSV, HIV, rabies virus,Hepatitis, and Respiratory Syncycial virus, and wherein said viraltransmembrane protein is selected from the group consisting ofglycoprotein E1, glycoprotein E2, and G protein.
 8. The geneticallyengineered membrane-enveloped virus of claim 1, wherein said viruses areRNA tumor viruses, and wherein said viral transmembrane protein is Env.9. A genetically engineered Sindbis virus comprising a viraltransmembrane glycoprotein that is able to span or correctly integrateinto the membrane of insect cells but not that of mammalian cells due todeletion of one or more amino acids in the transmembrane regions of saidviral transmembrane glycoprotein, wherein said virus is capable ofinfecting and producing progeny virus in insect cells, and is capable ofinfecting but not producing progeny virus in mammalian cells.
 10. Thegenetically engineered Sindbis virus of claim 9, wherein said viraltransmembrane protein is viral glycoprotein E2.
 11. The geneticallyengineered Sindbis virus of claim 9, wherein said virus is selected fromthe group consisting ofΔK391 virus, TM17 virus and TM16 virus.
 12. Amethod of producing a viral vaccine from the genetically engineeredmembrane-enveloped virus of claim 1 for vaccination of mammals,comprising the steps of: introducing said genetically engineeredmembrane-enveloped virus into insect cells; and allowing said virus toreplicate in said insect cells to produce a viral vaccine.
 13. Themethod of claim 12, wherein said genetically engineeredmembrane-enveloped virus is an Arthropod vectored virus.
 14. The methodof claim 12, wherein said virus is selected from the group consisting ofTogaviruses, Flaviviruses, Bunya viruses, enveloped viruses capable ofreplicating naturally in both mammalian and insect cells, and envelopedviruses which replicate in mammalian and insect cells as a result ofgenetic engineering of either the virus or the cell.
 15. A method ofproducing a viral vaccine from the genetically engineered Sindbis virusof claim 9 for vaccination of mammals, comprising the steps of:introducing said genetically engineered Sindbis virus into insect cells;and allowing said virus to replicate in said insect cells to produce aviral vaccine.
 16. The method of claim 15, wherein said Sindbis virus isselected from the group consisting of ΔK391 virus, TM17 virus and TM16virus.
 17. A method for vaccinating an individual in need of suchtreatment, comprising the steps of: introducing the viral vaccine ofclaim 12 into said individual; and allowing said viral vaccine toproduce viral proteins for immune surveillance and stimulate immunesystem for antibody production in said individual.
 18. A method forvaccinating an individual in need of such treatment, comprising thesteps of: introducing the viral vaccine of claim 15 into saidindividual; and allowing said viral vaccine to produce viral proteinsfor immune surveillance and stimulate immune system for antibodyproduction in said individual.
 19. A method of producing a viral vaccineto a disease spread by a mosquito population to a mammal, comprising thesteps of: producing deletions in the membrane associated domains ofviral transmembrane proteins in a membrane-enveloped virus, therebyrestricting the growth of said virus to insect cells; introducing saidmembrane-enveloped virus into a wild mosquito population; and allowingsaid membrane-enveloped virus to replicate in cells of said wildmosquito population to produce a mosquito population which excludes thewild type pathogenic virus and harbors the vaccine strain of saidmembrane-enveloped virus so that a mosquito bite delivers the vaccine tothe mammal bitten.
 20. The method of claim 19, wherein said deletions inthe membrane associated domains are prodcued by deleting one or moreamino acids in the transmembrane regions of said viral transmembraneproteins to produce transmembrane proteins that are able to span themembrane envelope of cells in said mosquito population, but are unableto span the membrane envelope of cells in said mammal, wherein saidmembrane-enveloped virus is capable of infecting and producing progenyvirus in said mosquito population, and is capable of infecting but notproducing progeny virus in mammalian cells.